Class IIb Microcin MccM Interferes with Oxidative Phosphorylation in Escherichia coli

Abstract

Dysbiosis of the human gut microbiota is linked to numerous diseases. Understanding the molecular mechanisms by which microbes interact and compete with one another is required for developing successful strategies to modulate the microbiome. The natural product Microcin M (MccM) consists of a 77-residue bioactive peptide conjugated to a siderophore and is a class II microcin involved in microbial competition with an enigmatic mode-of-action. In this work, we investigated the basis for MccM activity and leveraged bioinformatics to expand the known chemical diversity of class II microcins. We applied automated fast-flow solid phase peptide synthesis coupled with chemoenzymatic chemistry to acquire MccM and demonstrated that its activity was bacteriostatic. We then used our synthetic molecule to ascertain that catecholate siderophore transporters in Escherichia coli K-12 are necessary for MccM import. Once inside the cell, we found that MccM treatment decreased the levels of intracellular ATP and interfered with gene expression. These effects were ameliorated in genetic mutants lacking ATP synthase or in conditions that support substrate-level phosphorylation. Further, we showed that MccM elevated the levels of reactive oxygen species within the target cell. We propose that MccM effects its bacteriostatic activity by decreasing the total energy level of the cell through inhibition of oxidative phosphorylation. Lastly, using genome mining, we bioinformatically identified 171 novel putative class II microcins. Our investigation sheds light on the natural processes involved in microbial competition and provides inspiration, in the form of new molecules, for future therapeutic endeavors.

Figure 1.

Chemoenzymatic synthesis enables production of the class IIb microcin MccM. (a) Biosynthetic logic of class II microcins. (b) The primary sequence of the MccM precursor peptide. The leader region is italicized, and the core peptide (MccM peptide) is bolded. (c) The MGE post-translational modification of class IIb microcins is joined to the C-terminal serine of the MccM peptide. (d) The chemoenzymatic synthesis of MccM presented in this study.

Figure 2.

MccM antimicrobial activity against E. coli is dependent on the presence of the TBDRs FepA, Cir, and Fiu. (a) Activity of MccM against double and triple knock-out strains derived from E. coli H1443. (b) Activity of MccM against E. coli MG1655 and single knock-out strains from the Keio collection. (n = 3, sd)

Figure 3.

MccM is bacteriostatic and decreases cellular energy in E. coli. (a) Time-course study indicates that MccM activity is bacteriostatic. MccM or a negative control mixture was added to E. coli MG1655 at t = 0 h, and culture turbidity was monitored for 8 h. (b) MccM depletes ATP levels in the E. coli JW0334. MccM or a control was added to E. coli JW0334 at t = 0 min, and the intracellular ATP levels were monitored via an enzyme-coupled luminescence assay (Abcam). (c) MccM inhibits gene expression. The LacZ activity of E. coli JW0334 harboring pLacUV5_lacZ was measured using Miller’s assay. IPTG (2 mM) was added with MccM or the negative control mixture at t = 0 min. (n = 3, sd).

Figure 4.

Inactivation of F₁F_O-ATP synthase lessens the antimicrobial effects of MccM. (a) E. coli mutants lacking a functional ATP synthase grow to a higher optical density than a control strain with an intact ATP synthase when treated with 1 μM MccM. Compounds were added at t = 0, and culture turbidity after 16 h at 30 °C is shown. Full growth curves are plotted in Figure S5a. (b) MccM does not deplete ATP levels as much in mutants lacking a functional ATP synthase. Intracellular ATP levels were measured by a luminescence assay after 1 h of treatment with MccM. (c) Gene expression is less inhibited by MccM in strains lacking a functional ATP synthase. Gene expression was assayed via Miller’s assay on the gene product of lacZ expressed from the plasmid pAra_lacZ after 1 h. LacZ activity at both 0 and 1 h are plotted in Figure S5b. (n = 3, sd, all statistics were performed using one-way ANOVA analysis with a post-hoc Tukey’s HSD test)

Figure 5.

Increased access to substrates supporting substrate-level phosphorylation improves survival of E. coli when treated with MccM. Full antimicrobial activity curves plotted in Figure S6. (n = 3, sd, statistics were performed using one-way ANOVA analysis with a post-hoc Tukey’s HSD test)

Figure 6.

Genome mining reveals new class II microcins. (a) Sequence similarity network (E = 5) of known and newly discovered putative microcin precursor peptides. (b) Three newly discovered class IIb microcin biosynthetic gene clusters harboring unexpected biosynthetic enzymes.